Under our existing R21 grant, we developed a novel murine model of continuous particle infusion using an intramedullary femoral implant connected to an infusion pump in order to understand fundamental biological processes involved in wear particle-induced osteolysis. This clinically relevant model simulates the prosthetic environment more closely by using continuous delivery of particles to the bone-implant interface. The purpose of this R01 grant application is to elucidate the biological processes of chemokine-directed macrophage (MAC) and osteoprogenitor cell (OPC) trafficking to particles, using in vitro and in vivo models. This work will highlight the mechanisms of particle-induced MAC and OPC chemotaxis and trafficking, and suggest potential strategies and targets for mitigation of osteolysis. Specific Aim #1. To demonstrate that local MAC and OPC chemotaxis in vitro induced by particle-challenged MACs is mediated in part by two specific C-C chemokines: Macrophage Chemoattractant Protein-1 (MCP-1) and Macrophage Inflammatory Protein-1 alpha (MIP-11). Specific Aim #2. To demonstrate that MACs and OPCs introduced from a remote site will migrate to the femur in which continuously infused polyethylene particles are being delivered in vivo, using reporter genes and bioluminescence (BLI). Using [18F] fluoride ion Positron Emission Tomography (PET) scanning we will demonstrate a heightened local bone metabolic response to particle infusion. Specific Aim #3. To demonstrate that cell trafficking from remote sites to the particle-implant area in vivo is mediated by the chemokines MCP-1 and MIP-11. Interference with these chemokines and subsequent cell trafficking will blunt the foreign body reaction, decrease osteolysis, and decrease bone metabolic activity. Wild type murine MACs will be exposed to polyethylene particles in vitro. Local migration of wild type (CCR1++, CCR2++), CCR1-- (not responsive to MIP-11), and CCR2-- (not responsive to MCP-1) MACs and OPCs will be assessed quantitatively in a transwell cell migration apparatus in vitro. The effects of antibodies to MCP-1 and MIP-11 on cell chemotaxis will be tested. In vivo studies will use our murine model of continuous particle infusion, intramedullary femoral implant and infusion pump loaded with polyethylene particles. Reporter wild type, CCR1--, and CCR2-- MACs and OPCs will be injected via the tail vein of nude mice at day 6 post-operatively. Cell trafficking and metablic activity will be followed using sequential quantitative BLI and PET scanning at post-operative days 5 (one day before injection of cells on day 6), 7 , 8, 14, 21 and 28. MicroCT scans will document osteolysis. Additional studies will use infused chemokine antibodies, and chemokine knockout mice as the implant recipient. This research will elucidate important mechanisms of particle-induced, chemokine-directed cell trafficking and suggest new treatment strategies.